この作品は、DOE五大湖バイオエネルギー研究センター（DE-FC02-07ER64494 DOE科学BERオフィス）からの補助金によってサポートされていました。我々はプロトコルを完成させる上で有用な技術的な議論と貴重なコメントのための博士旭東張と彼のグループのメンバーに感謝しています。

1。サンプルの調製と酸抽出<ol><li>凍結乾燥した土壌サンプルのフィールドコレクションの後に。 </li><li>ボールミル、土壌グラインダー、または乳鉢と乳棒を用いて土壌試料を粉砕して均質化する。 </li><li> 25mLの加水分解フラスコに土壌サンプルを（&gt; 0.3 mgのNを含む）を量る。 </li><li>しっかりと、それぞれの加水分解フラスコに10mLの6M HClをフラスコにN 2ガ ​​スを充填し、キャップ。 </li><li>自動タイマースイッチを使用して8時間105℃インキュベーター内℃で加水分解する。 </li></ol> 2。サンプルの精製<ol><li>インキュベーターからフラスコを取り外し、室温まで冷却する。 </li><li>各フラスコに100μLの内部標準myo-イノシトール（水1mgを加え-1）を追加します。渦巻いて混和する。 </li><li> ST / NS 24 /安定性のためのプラスチック製コップに設定された40関節、200 mLの梨形フラスコに排出するプラスチック製の漏斗を設定します。 </li><li>倍ワットマン＃2定性的円（11センチ径）四半期及び漏斗にセットに。 </LI&gt; <li>スワール各加水分解フラスコを、フィルタする漏斗にスラリーを注ぎます（さらに水〜3 mLで各フラスコをすすぎがあります）。 </li><li> 〜45°C、適用する真空でロータリーエバポレーターを用いて濾液を乾燥させます。 </li><li> 3〜5 mLの水（必要に応じて超音波洗浄機を使用）で各梨フラスコから乾燥した残留物を再懸濁し、40 mLのテフロンチューブに注ぎ、水の第二のアリコートフラスコをすすいでください。 </li><li> 6.8沈殿金属イオンと他の有機分子に1M KOH溶液を使用して - 6.6にpHを調整します。 </li><li> 10分2000 RCFの遠心分離により沈殿物を除去。 </li><li> 40 mLのガラスチューブに上清を注ぐパラフィルムで真空管の開口部をカバーし、-20℃で凍結し、パラフィルムの穴を突く。 </li><li>すべての液体を除去するために凍結上清を凍結乾燥させます。 </li><li> 、3 mLの乾燥したメタノールで残渣を溶解する（必要に応じて超音波浴を使用して）徹底的にボルテックスし、キャップ、チューブ、そして解決するための10分間2000 rcfで遠心塩出。 </li><li> 3 mLの円錐形の反応バイアルに上澄みを移し、45°C（または必要に応じて乾燥した窒素ガスの穏やかな流れの下）で​​RapidVapマシンによって蒸発乾固する。 </li><li>ドライフリーズ100μL回復標準のN-メチルグルカミン（水1mgを加え-1）と1 mLのH 2 Oを追加し、各バイアルに、パラフィルムでバイアルの口をカバーし、-20℃で凍結、パラフィルムを穿孔し、 。 </li><li> 100μLムラミン酸（メタノールでは0.5 mg mL -1）を、100μLグルコサミン（水に1 mgのmL -1）を、100μLミオイノシトール（水1mgを加え-1）を追加、100μLN-：基準を作るメチル（水に1 mgのmL -1）を、1 mLのH 2 O、パラフィルム各バイアルは、-20℃で凍結し、凍結乾燥し、パラフィルムを穿孔。 </li></ol> 3。誘導体化<ol><li>は32 mg mL -1のヒドロキシルアミン塩酸塩および40 mg mL -1の 4 -ジメチルアミノピリジンを含む誘導体化試薬を準備します。ピリジン - メタノール（4:1 v / v）のピペリジンで。 </li><li>しっかりとキャップ、そして徹底的にボルテックス、reactivialsのそれぞれに誘導体化試薬を300μLを追加します。 </li><li> 35分75から80℃の水浴（よく、任意の水がバイアルを入力することができます避けるために、バイアルを密封）でバイアルを置きます。 </li><li>水浴から瓶を取り出し、室温まで冷却する。 </li><li> 3 mLのreactivials、しっかりとキャップ、徹底的に渦のそれぞれに1 mLの無水酢酸を追加し、75から80まで25分°Cの水浴で加熱。 </li><li>水浴から瓶を取り出し、室温まで冷却する。 </li></ol> 4。分離と測定<ol><li>各バイアル、しっかりとキャップ、徹底的にボルテックスに1.5 mLのジクロロメタンを追加します。 </li><li>ソリューションは、独立した二つのフレーズまで邪魔されずに座ってできるようにするために徹底的に各バイアル、しっかりとキャップと渦に1mLの1M HClを追加し、1000μLピペッターを使用して、上部（水性）相を吸引して捨てます。 </li><li>同じfのashionますが、1 mLのH 2 O（最後の洗浄工程と、水性トップフェーズのすべてが削除されていることを確認するために特別な世話をする）と、有機相を3回抽出する。 </li><li> （必要に応じて窒素ガスを使用するか、または乾燥）45 RapidVapを使用して、最終的な解決℃で乾燥させます。 </li><li> 300μL酢酸エチル - ヘキサン（1:1 v / v）でしっかりと少量の挿入とキャップ付き2 mLの琥珀色のスクリューキャップ付きバイアルへの転送に溶解する。 </li><li> 、定常フェーズ5％フェニル - 、95％メチルポリシロキサン（DB-5、または同等品）水素と長さ30 m、内径0.25 mm、0.25 umの膜厚：定量化のために、溶融シリカ極性キャピラリカラムを用いたGC-FIDで分析するまたは0.5 mLの分-1一定流量でキャリアガスとしてヘリウム、。クロマトグラフィーは、プレミアム &quot;不活性&quot;のフェーズと水素キャリアガスで良いですが、より一般的な品種で、ヘリウムと許容されます。検出器の設定は、300°Cであり、400 mLの分-1の空気と30 mLのmin -1の窒素の両方にと水素メークアップガス。注入し、オーブンのパラメータは次のとおりです。250℃に設定GCインレットと1μLスプリット注入（30:1）、初期オーブン温度、120℃、1分間保持、10℃オーブン温度を上げる℃以上。 -1〜250℃、2.5分間保持; 270ランプ°C 20°C min -1の 、2分を保持し、290ランプ°C 20°C min -1の 5分を保持します。キャリアガス流量を調整するので、250°Cにおけるイノシトール、グルコサミンとムラミン酸誘導体溶出、および270におけるN-メチルグルカミン溶出℃に</li></ol> 5。デリバティブ検証<ol><li>分子イオンを識別し、誘導体の分子量を決定するためにソフトなイオン化LC-ESI-TOF-MSを使用しています。 </li><li>デリバティブの対象となるイオンを調べるために感度のGC-EI-MS-SIMまたはGC-EI-MSMS強化を使用しています。 </li><li>誘導体の調製のために複数の同位体標識試薬を使用し、質量シフトを使用MSそれらの同位体標識誘導体の分子イオンとイオンの断片を調べた。 </li></ol> 6。代表的な結果酸分解、サンプル精製、誘導体化とGCの決定（ 図1）：メソッドのプロトコルは、主に4つのステップで構成されています。グルコサミンと標準株から土壌サンプルからムラミン酸の分析の例を図2に示されています。グルコサミンとムラミン酸、マンノサミンとガラクトサミン（グルコサミンの2種の異性体）のほかにも方法を用いて同時に決定することができます。内部標準ミオイノシトールに関する規格の応答の要因に基づいて、我々は土壌試料中のこれらのバイオマーカーを定量化することができます。回復基準は定性的に誘導体化のプロセスを監視するために使用されています。 aldononitrile酢酸誘導体、グルコサミンおよびムラミン酸の形成のためのスキームを図3に示されている</strong&gt;。 デリバティブの提案された構造は、感度の向上、またはソフトなイオン化LC-ESI-TOF-MS 8用GC-EIMS-SIMにより測定した。電子イオン化により形成されるイオンフラグメントの提案された構造は、イオンのスペクトルを比較することによって検討したサンプルは、さまざまな同位体incorporations 11で調製した。 図4は、非標識剤、D-酢酸無水、およびU -13 C-グルコサミンで調製した3つのシナリオにおける支配的なイオンaldononitrile酢酸のm / z 187の誘導体化グルコサミンの質量変化を示しています。その他の詳細情報および説明は、我々の最近の出版物8、11を参照することができます。この戦略は、デリバティブ化学を勉強するためのモデルとして役立つかもしれません。 <img alt="図1" src="/files/ftp_upload/3767/3767fig1.jpg" /> 土壌中のグルコサミンとムラミン酸の分析のためのプロトコルの図1測定のフローチャートサンプル。酸分解、精製、誘導体化とGC決定：プロトコルは、主に4つのステップが含まれています。 <img alt="図2" src="/files/ftp_upload/3767/3767fig2.jpg" /> 図2標準および土壌のイノシトール、グルコサミン、ムラミン酸、マンノサミン、ガラクトサミンとメチルのaldononitrile酢酸のGC-FIDクロマトグラム。 <img alt="図3" src="/files/ftp_upload/3767/3767fig3.jpg" /> 図3。aldononitrile酢酸誘導体、グルコサミンおよびムラミン酸の形成のためのスキーム。番号1は、ニトリルの反応を表しています。番号2は、アセチル化を表しています。 <a href="http://www.jove.com/files/ftp_upload/3767/3767fig4_lg.jpg"><img alt="図4" src="/files/ftp_upload/3767/3767fig4.jpg" /></a> 図4。支配的なイオンstructurに関連付けられているaldononitrile酢酸誘導体化グルコサミンのマススペクトル（A）非標識剤、（B）D-酢酸無水、（C）U -13 C-グルコサミンで調製したEIモードでのES。星は重い同位体原子または同位体基を表す。

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我々は、開示することは何もありません。

aldononitrile酢酸誘導体、グルコサミンおよびムラミン酸の分析のための提示GCベースの方法は、土壌から抽出されたこれらのアミノ糖を定量するため、比較的迅速な方法を提供する。誘導体は化学的に安定であり、1回の分析で決定することができます。メソッドは、土壌サンプルに限定されるものではなく、非土壌マトリックスからのサンプルに簡略化することができます。 <p class="jove_con...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ムラミン酸</td><td> Sigma-Aldrich社</td><td> M2503-25mgを</td></tr><tr><td> D-（+） - グルコサミン塩酸塩</td><td> Sigma-Aldrich社</td><td> G1514-100G </td></tr><tr><td> N-メチル-D-グルカミン</td><td> Sigma-Aldrich社</td><td> M2004-500G </td></tr><tr><td>ミオイノシトール</td><td>フィッシャー·サイエンティフィック</td><td> A307003G025 </td></tr><tr><td>メタノール（ドライ） </td><td>アクロスの有機物</td><td> AC3269500​​10 </td></tr><tr><td> 4 - ジメチルアミノピリジン</td><td>アクロスの有機物</td><td> AC148270050 </td></tr><tr><td>酢酸エチル</td><td> VWRインターナショナル</td><td> BJGC100-4 </td></tr><tr><td> Hydroxlamine塩酸塩</td><td>フィッシャー·サイエンティフィック</td><td> H330-100 </td></tr><tr><td>ピリジン</td><td>フィッシャー·サイエンティフィック</td><td> P368-500 </td></tr><tr><td>無水酢酸</td> &lt;TD&gt;フィッシャー·サイエンティフィック</td><td> A10-100 </td></tr><tr><td>ジクロロメタン（塩化メチレン） </td><td>フィッシャー·サイエンティフィック</td><td> D37-500 </td></tr><tr><td>ヘキサン</td><td>フィッシャー·サイエンティフィック</td><td> H303-4 </td></tr><tr><td>塩酸（6M） </td><td>フィッシャー·サイエンティフィック</td><td> S456-4 </td></tr><tr><td>ヒドロキシルアミン塩酸塩-15 N </td><td>アイコンサービス</td><td> IN5280 </td></tr><tr><td>無水酢酸-2 H（D 6 C 4 O 3） </td><td>アクロスの有機物</td><td> AC174670050 </td></tr><tr><td> D-グルコース-U -13 C </td><td>ケンブリッジアイソトープ実験室</td><td> CLM-1396から1 </td></tr><tr><td>アンモニウムsufate -15 N </td><td>ケンブリッジアイソトープ実験室</td><td> NLM-713から1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 機器の名前 </td><td> 会社 </td><td> タイプ </td></tr><tr><td>ラピッドバップ</td&gt; <td> LabConco </td><td> 790002 </td></tr><tr><td> Vacumポンプ</td><td> KNF Neuberger </td><td> D-79112 </td></tr><tr><td>加水分解フラスコ</td><td>フィッシャー·サイエンティフィック</td><td> 06 423A </td></tr><tr><td>マイクロバイアル誘導体化</td><td>フィッシャー·サイエンティフィック</td><td> 06-100E </td></tr><tr><td> GC </td><td>ヒューレット·パッカード</td><td> 6890 </td></tr><tr><td> MS </td><td>ヒューレット·パッカード</td><td> 5972 </td></tr><tr><td> LC-ESI-TOF-MS </td><td>アジレント</td><td> Agilent 1200シリーズHPLCシステムは、Agilent LC / MSD-TOFに結合</td></tr></tbody></table>

gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil

微生物を特徴付けるに定量的なアプローチは、生態系内の微生物の状態や機能の広範な理解のために重要である。微生物の分析のための現在の戦略は、従来の研究室文化に依存する技術と直接抽出し、特定のバイオマーカー1、2の決定に基づいて、それらの両方が含まれます。土壌に生息する微生物種の多様性の間でいくつかを培養し、その文化に依存する方法は重要なバイアス、バイオマーカー分析ではない制限を導入することができます。 これらのグルコサミン（最も豊富に存在する）と、ムラミン酸のグルコサミン、マンノサミン、ガラクトサミンとムラミン酸だけでなく生者と死者の微生物の質量の両方の対策として供されているが、（一意細菌細胞から）3土壌システムで最も重要な成分である、4。しかし、分析に関する知識の欠如は、科学的なピア間の幅広い普及を制限します。すべてのEXIの間で分析方法刺す、GCベースの分析に続いてaldononitrileに誘導体化アセテートは良い最適のバランス精度に関してオプション性、感度、シンプルさ、優れたクロマトグラフ分離、およびサンプル·ストレージ·5時の安定性として浮上している。 ここでは、aldononitrile酢酸への変換後の土壌からのグルコサミンとムラミン酸の信頼性が高く、比較的単純な分析のための詳細なプロトコルを提示します。酸分解、サンプル精製、誘導体化とGC決定：プロトコルは、主に4つのステップで構成されています。ステップバイステップの手順は、前者の刊行物6,7に応じて変更されています。加えて、我々は電子イオン化により形成される構造誘導体の分子イオンを検証するための戦略とそのイオンの断片を提示します。我々はaldononitrile酢酸誘導体、グルコサミンとMURの分子量を決定するためにGC-EI-MS-SIM、LC-ESI-TOF-MSおよび同位体標識試薬を適用しアミック酸、我々は、各デリバティブ8のイオンフラグメントを調べるためのイオンスペクトルの同位体で標識した誘導体の質量シフトを使用していました。理論的な解明に加えて、分子の誘導体のイオンとそのイオンフラグメントの検証では、生物地球化学的研究9、10に、これらのバイオマーカーのδ13 Cまたはイオンフラグメントを使用して研究者に有用であろう。

Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

我々は、グルコサミンや土壌から抽出されたムラミン酸のaldonitrile酢酸誘導体のGC-ベースの分析のためのメソッドがプロトコルを記述します。化学的機構の解明のために、我々はまた、デリバティブおよび電子イオン化により形成されるイオンフラグメントの構造を確認するための戦略を提示します。

土壌中の微生物の残留決定のためのAldononitrile酢酸誘導体化グルコサミンとムラミン酸のGCベースの検出

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

gc-based-detection-aldononitrile-acetate-derivatized-glucosamine

Research

JoVE Journal

Biology

16.8K ビュー.  University of Wisconsin, Madison. 我々は、グルコサミンや土壌から抽出されたムラミン酸のaldonitrile酢酸誘導体のGC-ベースの分析のためのメソッドがプロトコルを記述します。化学的機構の解明のために、我々はまた、デリバティブおよび電子イオン化により形成されるイオンフラグメントの構造を確認するための戦略を提示します。

ビデオ: GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Microbial Communities in Nature and Laboratory - Interview

自然と実験室での微生物群集 - インタビュー

生物学

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

酵素酢酸キナーゼのアセテート形成活性の直接検出

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

微生物システム生物学のケモスタットの使用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

農業で使用する生分解性プラスチックマルチフィルムを分解するための可能性を持つネイティブ土壌微生物の分離

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

微生物の同定とキャラクタリゼーションのためのパイロシーケンシング

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

マウス初代肝細胞における脂肪酸酸化および脂質生成の決意

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

enChIPによって特定のゲノム領域と関連する分子の同定の単離

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

単離されたワーキングラット心臓におけるグルコースおよび脂肪酸酸化の宿泊料金を決意するための方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

病抑制のクイックの予備評価として板競争の試金

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.